摘要
目的探讨巨噬细胞外泌体源miR-223对胃癌细胞转移能力的影响及作用机制。方法选择巨噬细胞及其外泌体分别与胃癌细胞共培养(未加为对照组),检测miR-223表达量并观察其对胃癌细胞转移能力的影响。荧光显微镜观察巨噬细胞是否通过外泌体传递miR-223至胃癌细胞。巨噬细胞转染miR-223拮抗剂,分离外泌体与胃癌细胞共培养,transwell、划痕实验观察其对胃癌细胞转移的影响,逆转录聚合酶链反应(RT-PCR)及Western印迹检测蛋白酪氨酸磷酸酶(PTEN)及转移相关蛋白表达。结果巨噬细胞及其外泌体与胃癌细胞共培养后,胃癌细胞迁移及侵袭能力增强[253.2±6.3(对照组)、451.8±12.8、453.4±14.4,与对照组比较均P<0.01;98.4±5.1(对照组)、276.5±10.3、257.3±8.5,与对照组比较均P<0.01],miR-223(1.00±0.00、8.83±0.91、9.57±1.03)相对表达量差异有统计学意义(均P<0.05)。荧光显微镜观察显示巨噬细胞通过外泌体传递miR-223至胃癌细胞。阻断巨噬细胞miR-223表达后,其来源外泌体促进胃癌细胞迁移和侵袭的能力明显降低(215.6±9.2、402.5±11.6、253.7±10.4,均P<0.01;91.5±8.2、263.4±9.3、105.8±9.3,均P<0.01)。胃癌细胞与巨噬细胞来源外泌体共培养后PTEN mRNA表达下降(1.00±0.00比0.26±0.03),相对表达量差异有统计学意义(P<0.05);PI3K/AKT通路激活,细胞骨架重塑。阻断miR-223传递后这一激活作用减弱。结论巨噬细胞通过外泌体传递miR-223至胃癌细胞,靶向PTEN并激活PI3K/AKT信号通路从而促进胃癌细胞的转移。
Objective To investigate the effect and mechanism of exosome-derived miR-223 from macrophage on gastric cancer(GC)cell metastasis.Methods Exosomes isolated from macrophages culture medium were characterized and cocultured with GC cell,the miRNA level was detected by qRT-PCR.The migration and invasion of GC cell were detected by transwell.The internalization of exosomes,transfer of miR-223 was observed by immunofluorescence.Macrophage were transfected with a miR-223 inhibitor or negative control,transwell and scratch test were employed to explore the effect of macrophage derived exosome on the migration and invasion of GC cell.Western blot and RT-PCR assay were performed to uncover the underlying mechanisms of miR-223 and PTEN-PI3K/AKT pathway.Results This study showed that macrophage and macrophage-derived exosomes promoted the migration and invasion of gastric cancer cell(253.2±6.3,451.8±12.8,453.4±14.4,all P<0.01,and 98.4±5.1,276.5±10.3,257.3±8.5,all P<0.01,respectively).miR-223 was enriched in macrophage-derived exosomes,which was transferred to the co-cultivated gastric cancer cells.miR-223 knockdown in macrophage reversed the migration and invasion of exosomes on gastric cancer cells(215.6±9.2,402.5±11.6,253.7±10.4,all P<0.01,and 91.5±8.2,263.4±9.3,105.8±9.3,all P<0.01,respectively).Functional studies revealed that exosomal miR-223 derived from macrophage promoted the metastasis of GC cells via the PTEN-PI3K/AKT pathway.In addition,itshowed thatthe actin cytoskeleton was altered,and multiple proteins associated with epithelial-mesenchymaltransition(EMT)were upregulated.Conclusion Exosomal transfer of macrophage-derived miR-223 promote the metastasis of GC cells through targeting the PTEN-PI3K/AKT pathway.
作者
郑培明
高慧洁
李俊蒙
张鹏
李刚
Zheng Peiming;Gao Huijie;Li Junmeng;Zhang Peng;Li Gang(Department of Clinical Laboratory,Henan Provincial People′s Hospital,Zhengzhou University People′s Hospital,Henan University People′s Hospital,Zhengzhou 450003,China;Department of Oncology,the First Affiliated Hospital of Henan University,Kaifeng 475000,China;Department of Gastrointestinal surgery,Henan Provincial People′s Hospital,Zhengzhou University People′s Hospital,Henan University People′s Hospital,Zhengzhou 450003,China)
出处
《中华医学杂志》
CAS
CSCD
北大核心
2020年第22期1750-1755,共6页
National Medical Journal of China
基金
国家自然科学基金青年项目(81802094)。
